It has now been demonstrated that tissues of many plant species may be transformed by exogenous, typically chimeric, genes which are effective to stably transform cells of the tissues. For several species, tissues transformed in this fashion may be regenerated to give rise to whole transgenic or genetically engineered plants. The engineered traits introduced into the transgenic plants by these techniques have proven to be stable and have also proven to be transmissible through normal Mendellian inheritance to the progeny of the regenerated plants. In those species in which the ability to construct transgenic plants has been established and replicated, such as in tobacco, much research focus is logically directed next toward the introduction of useful traits into those plants. One such desirable trait is the production in the plant cells of desired gene products in vivo in the cells of the transgenic plants.
The most common, though by no means unique, method of transformation of plant cells used to date is based on a unique property of the plant pathogen Agrobacterium tumefaciens. Natural or wild-type A. tumefaciens, in its normal pathogenic process, transmits a portion of a Ti (for Tumor-inducing) plasmid that it harbors to be introduced into the genome of the infected plant host. This portion of the Ti plasmid is referred to as the T-DNA. The Agrobacterium performs this pathogenic transformation in nature to direct the host cells of the plant to become tumorous and to produce a class of plant metabolites called opines on which the Agrobacterium has the unique ability to feed. By removing the genes responsible for tumor induction and opine production from the Ti plasmid, and by substituting for them exogenous chimeric genes of interest, the plant genetic engineer may then use the natural pathogenic process of the A. tumefaciens to introduce foreign genes into plant tissues. Because this transformation will generally occur only on somatic plant tissues which have been wounded, its use to date has focused on those species, such as tobacco, which can be regenerated either from individual somatic cells or from embryogenic somatic cell cultures. This technique has proved effective for plant transformations in cotton, tomato, carrot, and petunia, as well as some other species.
Other plant cell transformation techniques are directed toward the direct insertion of DNA into the cytoplasm of plant cells from which it is taken up, by an uncharacterized mechanism, into the genome of the plant. One such technique is electroporation, in which electric shock causes disruption of the cellular membranes of individual plant cells. Plant protoplasts in aqueous solution when subject to electroporation will uptake DNA from the surrounding medium. Another technique involves the physical acceleration of DNA, coated onto small inert particles, either into regenerable plant tissues or into plant germline cells. These techniques widen the range of plant species which may be genetically engineered since they allow for the transformation of a wider variety of tissue types such as embryonic tissues, or germline cells.
Having the ability to introduce foreign DNA constructs into the genome of plants, however, does not in and of itself create useful traits in the modified plants or plant lines. The ability to code for the production of proteins in plant cells can only contribute to making a more useful plant or plant line if the protein offers some advantage in the field to the plant and is produced in the plant cells in quantities effective to accomplish the desired objective. One objective in the creation of transgenic plants is to make plants which are less attractive to potential plant predators or pathogens. A candidate strategy to make plants resistant to certain insect predators is based on a unique protein made by the Bacillus thuringiensis, known as the delta-endotoxin or crystal protein. While the various B. thuringiensis species have relatively large variations in the DNA coding sequences for their delta-endotoxin proteins, the proteins themselves have a relatively high degree of homology. This toxin is a relatively large protein that has a specific toxicity to Lepidopteran, Dipteran, or Coleopteran insects. While insecticidal peptides made by the Bacillus thuringiensis (B.t.) species have been approved for use, and have been used, in agriculture for many years, the relatively high cost of producing the protein in quantity and the need for repeated applications of the protein, because of its degradation in the environment, have proved to be limits on the extensive use of these materials. The creation of transgenic plants which generate this biological insecticide by themselves offers a practical mechanism to control susceptible insects without the need for repeated application of other control agents.
A primary target species for the introduction of an effective B.t. toxin capability is the crop plant cotton (Gossypium hirsutum L.) In the United States, cotton is an agricultural crop with an exceptionally high pesticide requirement, and that requirement often includes formulations of Bt. toxin produced by bacteria. The Lepidopteran pests of cotton include the tobacco budworm (Heliothis virescens), the corn earworm (Heliothis zea), also called the cotton bollworm, and the beet armyworm (Spodoptera frugiperda). Because of the long regeneration time required to regenerate whole cotton plants from transformed tissues, however, it is practical to use tobacco as a model species to demonstrate and test vector and gene constructions and expression strategies. The inventors here have previously demonstrated the ability to adapt transformation and expression techniques from tobacco to the successful transformation and regeneration of cotton plants and lines. Umbeck et al., "Genetically Transformed Cotton (Gossypium hirsutum L.) Plants," Bio/Technology, 5, pp 263-266 (1987).
Another consideration in the genetic transformation of plants to express useful proteins is the method of construction of appropriate chimeric DNA sequences which are practically effective to achieve practical transcription and translation levels of the foreign gene products in plant cells. To be effective, a foreign DNA sequence containing a coding region must be flanked by appropriate promotion and control regions. Commonly used plant cell transcription promoters include the nopaline synthase promoter from the T-DNA of A. tumefaciens and the 35S promoter from the cauliflower mosaic virus. These promoters are effective in most plant cells but the level of transcription and translation activities of protein coding sequences placed down stream of these promoters is quite variable, depending on several factors such as insertion site or sites and copy number of insertions. Other variables, such as untranslated portions of the transcription product and the polyadenylation sequence also effect the level of translational activity of the coded gene product.
Specifically with regard to the crystal protein of Bacillus thuringiensis, it has been previously demonstrated that the crystal protein itself consists of one or more species of a large protein up to 160 kilodaltons in size. This large protein is now referred to as a protoxin, since it has been determined that the protoxin may be cleaved by proteolysis (and is so cleaved in the insect gut) to produce an active peptide toxin of a molecular weight of 55 to 75 kilodaltons that retains the specific toxicity to the target insects. Deletion analysis has localized the toxic portion of the protoxin to the amino terminal end of the protoxin and have demonstrated that both amino- and carboxy-terminal fusions can be made to the toxin without loss of insecticidal activity. The function of the remaining carboxyl portions of the protoxin, beyond structural considerations in crystal protein formation, remains unknown.
While expression of several model proteins in model plant species has proved a regularly replicable process, some proteins present special problems. The B.t. protoxin molecule is very large and quite insoluble. The expression of this protein in regenerated transgenic plants has proven to be difficult. The coding sequence for the protein can reliably be inserted into normally competent plant transformation and expression vectors, but the recovery and regeneration of expressing tissues is difficult. Tissues in culture in which the entire protoxin is expressed can be created, but these tissues are typically necrotic or visibly unhealthy and cannot routinely be regenerated into whole plants. This observation may be due to toxic effects of the protoxin or perhaps simply by its insolubility.